CN109010422A - A kind of preparation method and applications of Qingqian Willow leaf general flavone - Google Patents

Abstract

The invention belongs to the field of Chinese medicines, and in particular to a kind of preparation method of Qingqian Willow leaf general flavone and its application in the drug and health care product of preparation prevention and treatment nonalcoholic steatohepatitis.The preparation method of Qingqian Willow leaf general flavone provided by the invention, using dry Qingqian Willow leaf, through the obtained Qingqian Willow leaf extracting solution such as crushing, extraction, filtering, it is extracted using double-aqueous phase system, Qingqian Willow leaf general flavone solution is in glassy yellow, it is enriched in phase, further separates, depressurizes, is concentrated, is drying to obtain.80%-95% is up to using Qingqian Willow leaf general flavone extraction yield produced by the present invention.Qingqian Willow leaf general flavone prepared by the present invention has reduction transaminase activity, and hypoglycemic, reducing blood lipid inhibits the effect of accumulation of fat, has ensured application of the Qingqian Willow leaf general flavone in preparation treatment nonalcoholic steatohepatitis drug.

Description

A preparation method and application of total flavonoids from Cyclocarya paliurus leaves

technical field

The invention belongs to the field of traditional Chinese medicines, and in particular relates to a preparation method and application of total flavonoids from leaves of Cyclocarya paliurus.

Background technique

Non-alcoholic steatohepatitis (NASH) refers to a type of chronic hepatitis with liver histopathological changes similar to alcoholic hepatitis, but without a clear history of drinking. It is derived from excessive accumulation of liver fat associated with insulin resistance (IR) and has hepatocyte degeneration , necrosis, inflammation and infiltration and other characteristics, will further develop into liver cirrhosis and liver cancer. The incidence of NASH is increasing with the development of the economy. About 30% of the obese people will develop NASH. According to the statistics of WHO in 2016, more than 650 million people in the world are obese. NASH has become a threat to human health. serious problem. The "two-hit" theory caused by IR is the main pathogenesis of NASH: the first hit is hepatic fat deposition and liver cell degeneration caused by IR; the second hit is oxidative stress and lipid peroxidation damage, causing liver cell Chronic persistent damage and inflammation of itself and mitochondria.

Screening effective drugs based on the pathogenesis of NASH is a current research focus, focusing on insulin sensitizers, antioxidants, and lipid-lowering drugs. Chinese patent application CN105592846 A discloses a method for treating fatty liver disease or disease in subjects in need. Although the method has a certain inhibitory effect on NASH, the range of subjects is small, the treatment period is long, and The used pharmaceutical composition contains substances such as amides and enoic acids, which have certain toxic and side effects.

Cyclocarya paliurus is a deciduous tree of the genus Cyclocarya paliurus in the family Juglandaceae. The leaves taste sweet and have the effects of promoting body fluid and clearing away heat, lowering blood pressure, lowering blood sugar, lowering blood fat, and prolonging life. "China Traditional Chinese Medicine Resources Chronicle" records that the leaves and bark of Cyclocarya paliurus have the functions of clearing away heat, reducing swelling and relieving pain. Cyclocarya paliurus leaves mainly contain polysaccharides, flavonoids, and triterpenoids and other chemical components. Studies on pharmacological activity have shown that polysaccharides in Cyclocarya paliurus have pharmacological activities such as lowering blood sugar, lowering blood fat, and anti-lipid peroxidation; flavonoids can inhibit the activity of α-glucosidase and have a strong ability to scavenge free radicals; At present, no research has been found on the treatment of NASH with the total flavonoids of Cyclocarya paliurus leaves.

Contents of the invention

Aiming at the defects in the prior art, the object of the present invention is to provide a preparation method and application of total flavonoids from Cyclocarya paliurus leaves. In order to achieve the above object, the invention provides a method for preparing total flavonoids of Cyclocarya paliurus leaves, the specific operation steps are as follows:

a. Take the dried leaves of Cyclocarya paliurus, crush them, and pass through a 40-50 mesh sieve to obtain Cyclocarya coarse powder. Soak Cyclocarya paliurus coarse powder in aqueous ethanol for 0.5-1.5 hours, then heat and reflux to extract 1- 3h, after a total of 3-5 extractions, filter and combine to obtain Cyclocarya paliurus leaf extract;

b. adding the Cyclocarya paliurus leaf extract prepared in step a to polyethylene glycol and phosphate buffer to form a two-phase system;

c. Adjust the pH of the two-phase system to 6.5-7.5, stir it magnetically for 10 minutes, and then let it stand at 25°C for 1-2 hours. The total flavonoids of Cyclocarya paliurus leaves are enriched in the upper phase, showing bright yellow. The mixture of total flavonoids solution of Cyclocarya paliurus leaves and polyethylene glycol;

d. Pipette the upper layer mixture obtained in step c, put it on a D101 macroporous resin column, elute with ultrapure water to remove polyethylene glycol, and then elute with ethanol aqueous solution to obtain the eluate of total flavonoids of Cyclocarya paliurus leaves;

e. further depressurizing, concentrating and drying the eluate of total flavonoids from Cyclocarya paliurus leaves obtained in step d.

Preferably, the mass of the ethanol aqueous solution in step a is 5-15 times the mass of Cyclocarya paliurus coarse powder, and the volume fraction of the ethanol aqueous solution is 55-95%.

Preferably, the concentration range of polyethylene glycol in the step b is 15%-25%; the concentration range of the phosphate buffer is 15%-20%.

Preferably, the concentration of the total flavonoids solution of Cyclocarya paliurus leaves in the step c is 50 mg/mL.

Preferably, in the D101 macroporous resin column of the step d, the volume ratio of the loading volume to the D101 macroporous resin is 1:15, and the volume of the ultrapure water is 20% of the volume of the D101 macroporous resin column. times, the concentration of the aqueous ethanol solution is 80%, and the volume is 15 times the volume of the D101 macroporous resin column.

Compared with the prior art, the present invention has the advantages of:

(1) The total flavonoids of Cyclocarya paliurus leaf provided by the present invention has simple preparation method, rich raw materials, heating and reflux extraction, increased solvent penetration, high extraction rate, and short extraction time, thereby realizing efficient and rapid preparation of Cyclocarya paliurus leaf total flavonoids The purpose of flavonoids;

(2) The present invention adopts two-phase extraction method to extract flavonoids, and two-phase extraction method is an efficient and gentle separation technology, which is easy to amplify and maintain the biological activity of active ingredients;

(3) the total flavonoids of Cyclocarya paliurus leaf that the present invention extracts has effect such as reducing transaminase, reducing blood fat, hypoglycemia, reducing lipid peroxidation, suppressing liver cell fibrosis, can be effectively used in the preparation of the medicine for preventing and treating nonalcoholic steatohepatitis The new use provides a great boost to the realization of a healthy China.

Description of drawings

Fig. 1 is the influence of total flavonoids of Cyclocarya paliurus leaf on α-glucosidase activity;

Figure 2 is the effect of total flavonoids from Cyclocarya paliurus leaves on the organ indexes of NASH model mice;

Fig. 3 is the influence of the total flavonoids of Cyclocarya paliurus leaves on the biochemical indicators of NASH model mice;

Figure 4 is the effect of total flavonoids from Cyclocarya paliurus leaves on the liver morphology of NASH model mice;

Figure 5 shows the effect of total flavonoids from Cyclocarya paliurus leaves on liver pathology in NASH model mice.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples, but the following examples are not intended to limit the present invention, and all methods or technical solutions close to the present invention are in within the protection scope of the present invention.

Embodiment 1 A kind of preparation method of total flavonoids of Cyclocarya paliurus leaves

The preparation method of the total flavonoids of the leaves of Cyclocarya paliurus is as follows:

a. Get the dry leaves of Cyclocarya paliurus, pulverize, cross 40 mesh sieves to get Cyclocarya coarse powder, put Cyclocarya paliurus coarse powder into 5 times of the quality of Cyclocarya paliurus coarse powder, and be 55% ethanol by volume fraction Soak in the aqueous solution for 0.5h, then heat and reflux for extraction for 1h, extract 3 times in total, filter and combine to obtain Cyclocarya paliurus leaf extract;

b. add the Cyclocarya paliurus leaf extract prepared in step a to polyethylene glycol and phosphate buffer to form a two-phase system, wherein the mass concentration of polyethylene glycol is 15%, and the mass concentration of phosphate buffer is The concentration is 15%; the phosphate buffer solution is prepared by dissolving 34.0 g of anhydrous potassium dihydrogen phosphate (KH ₂ PO ₄ ) and 35.5 g of anhydrous disodium hydrogen phosphate (Na ₂ HPO ₄ ) in water and diluting to 1000 mL to make;

c. Adjust the pH value of the two-phase system to 6.5, stir it magnetically for 10 minutes, and then let it stand at 25°C for 1 hour. The total flavonoids of the leaves of Cyclocarya paliurus are enriched in the upper phase, showing bright yellow, and the leaves of Cyclocarya paliurus are obtained in the upper layer. A mixture of total flavonoids solution and polyethylene glycol, the concentration of the total flavones solution of Cyclocarya paliurus leaves is 50mg/mL;

d. Pipette the mixture obtained in step c on the D101 macroporous resin column, the ratio of the loading amount to the resin volume is 1: 15, and remove polyethylene glycol by elution with ultrapure water of 20 times the volume of the D101 macroporous resin column, Then elute with 80% ethanol aqueous solution of 15 times the volume of the D101 macroporous resin column to obtain the eluate of total flavonoids of Cyclocarya paliurus leaves;

e. further depressurizing, concentrating and drying the eluate of total flavonoids from Cyclocarya paliurus leaves obtained in step d.

The extraction rate of the total flavonoids from the leaves of Cyclocarya paliurus is 85%.

Embodiment 2 A kind of preparation method of total flavonoids of Cyclocarya paliurus leaves

The preparation method of the total flavonoids of the leaves of Cyclocarya paliurus is as follows:

a. Get the dried leaves of Cyclocarya paliurus, pulverize them, and cross a 50-mesh sieve to obtain Cyclocarya coarse powder, which is 95% ethanol with 15 times the mass of Cyclocarya paliurus coarse powder. Soak in aqueous solution for 1.5h, then heat and reflux for extraction for 3h, extract 5 times in total, filter and combine to obtain Cyclocarya paliurus leaf extract;

b. add the Cyclocarya paliurus leaf extract prepared in step a to polyethylene glycol and phosphate buffer to form a two-phase system, wherein the mass concentration of polyethylene glycol is 25%, and the mass concentration of phosphate buffer is The concentration is 20%. The phosphate buffer solution is prepared by dissolving 34.0 g of anhydrous potassium dihydrogen phosphate (KH ₂ PO ₄ ) and 35.5 g of anhydrous disodium hydrogen phosphate (Na ₂ HPO ₄ ) in water and diluting to 1000 mL. to make;

c. Adjust the pH value of the two-phase system to 7.5, stir magnetically for 10 minutes, and then let it stand at 25°C for 2 hours. The total flavonoids of Cyclocarya paliurus leaves are enriched in the upper phase, showing bright yellow color, and the top layer is Cyclocarya paliurus leaves. A mixture of total flavonoids solution and polyethylene glycol, the concentration of the total flavones solution of Cyclocarya paliurus leaves is 50mg/mL;

d. Pipette the mixture obtained in step c on the D101 macroporous resin column, the ratio of the loading amount to the resin volume is 1: 15, and remove polyethylene glycol by elution with ultrapure water of 20 times the volume of the D101 macroporous resin column, Then elute with 80% ethanol with 15 times the volume of the D101 macroporous resin column to obtain the eluate of total flavonoids of Cyclocarya paliurus leaves;

e. further depressurizing, concentrating and drying the eluate of total flavonoids from Cyclocarya paliurus leaves obtained in step d.

The extraction rate of the total flavonoids from the leaves of Cyclocarya paliurus is 87%.

Embodiment 3 A kind of preparation method of total flavonoids of Cyclocarya paliurus leaves

The preparation method of the total flavonoids of the leaves of Cyclocarya paliurus is as follows:

a. Get the dried leaves of Cyclocarya paliurus, pulverize them, and cross a 45 mesh sieve to get Cyclocarya coarse powder, place Cyclocarya paliurus coarse powder in 10 times of the quality of Cyclocarya paliurus coarse powder, and have a volume fraction of 75% ethanol Soak in the aqueous solution for 1 hour, then heat and reflux for extraction for 2 hours, extract 4 times in total, filter and combine to obtain Cyclocarya paliurus leaf extract;

b. add the Cyclocarya paliurus leaf extract that step a makes in polyethylene glycol and phosphate buffered saline, form two-phase system, wherein, the concentration of polyethylene glycol is 20%, the concentration of phosphate buffered saline is 17%, the phosphate buffer solution is prepared by dissolving 34.0 g of anhydrous potassium dihydrogen phosphate (KH ₂ PO ₄ ) and 35.5 g of anhydrous disodium hydrogen phosphate (Na ₂ HPO ₄ ) in water and diluting to 1000 mL;

c. Adjust the pH value of the two-phase aqueous system to 7.0, stir magnetically for 10 minutes, and then let it stand at 25°C for 1.5 hours. The total flavonoids of Cyclocarya paliurus leaves are enriched in the upper phase, showing bright yellow, and the upper layer and the upper layer are green money. A mixture of willow leaf total flavonoids solution and polyethylene glycol, the concentration of the total flavonoids solution in Cyclocarya paliurus leaves is 50mg/mL;

d. pipette the mixture obtained in step c on the D101 macroporous resin column, the ratio of the sample volume to the resin volume is 1: 15, and remove polyethylene glycol and buffer by elution with ultrapure water of 20 times the volume of the D101 macroporous resin column salt, and then eluted with 80% ethanol with 15 times the volume of D101 macroporous resin column to obtain the eluate of total flavonoids of Cyclocarya paliurus leaves;

e. further depressurizing, concentrating and drying the eluate of total flavonoids from Cyclocarya paliurus leaves obtained in step d.

The extraction rate of the total flavonoids from the leaves of Cyclocarya paliurus is 95%.

Test example 1 Inhibitory effect of total flavonoids of Cyclocarya paliurus leaves on α-glucosidase

1. Test sample: the total flavonoids of Cyclocarya paliurus leaves prepared by the method described in Example 3.

2. Test method: In a 96-well plate, add 20 μL α-glucosidase solution (PBS, pH6.8, 1U/mL), 50 μL positive control drug acarbose (10, 20, 40, 80, 160 , 320μM) and different concentrations of total flavonoids from Cyclocarya paliurus leaves (5, 10, 20, 40, 80, 160, 320μg/mL) solution, incubated at 37℃ for 15min, then added 20μL p-NPG (5mM) solution, incubated at 37℃ 20min, add 80μL Na ₂ CO ₃ (0.1M) to terminate the reaction, immediately use a microplate reader to measure the absorbance value at 405nm, without adding the solution of total flavonoids of Cyclocarya paliurus leaves as a blank control, calculate the effect of each component on α-glucoside Enzyme inhibition rate. Set up 4 duplicate wells for each sample, take the average value, and calculate the IC ₅₀ value of the total flavonoids of Cyclocarya paliurus leaves on α-glucosidase.

The formula for the inhibition rate of the sample to α-glucosidase activity is as follows: inhibition rate%=(A _control -A _sample )/(A _control -A _blank )×100%. (A _blank is the absorbance value of the blank control well; A _control is the absorbance value of the well without inhibitor; A _sample is the absorbance value of the sample well).

3. Test results: The total flavonoids of Cyclocarya paliurus leaves can dose-dependently inhibit the activity of α-glucosidase in the dose range of 20μg/mL-320μg/mL, and its IC ₅₀ is 151.40±4.92μg/mL, the results are shown in the figure 1. It shows that the total flavonoids of Cyclocarya paliurus leaves can lower blood sugar by inhibiting the activity of α-glucosidase. Therefore, it is effective for non-alcoholic steatohepatitis caused by high blood sugar level, or high blood sugar complicated by non-alcoholic steatohepatitis. have a good intervention effect.

Test Example 2 Effect of total flavonoids from Cyclocarya paliurus leaves on NASH mouse model induced by high-fat diet

1. Test sample: the total flavonoids of Cyclocarya paliurus leaves prepared by the method described in Example 3.

2. Experimental animals: Clean grade C57BL/6 mice were purchased from Guangdong Medical Experimental Animal Center, animal license number SCXK (Guangdong) 2013-0002.

3. Test reagents and instruments: Cyclocarya paliurus leaves, identified as dry leaves of Cyclocarya paliurus, purchased from Suichang, Zhejiang; Metformin hydrochloride tablets, purchased from Sino-US Shanghai Bristol-Myers Squibb Pharmaceutical Co., Ltd.; Acarbose tablets, purchased from Bayer Medicine and Health Care Co., Ltd.; α-glucosidase (Roche subpackage) was purchased from Dalian Meilun Biotechnology Co., Ltd.; 4-nitrophenol-α-D-glucopyranoside (p-NPG) was purchased from Tsi A Shanghai) Chemical Industry Development Co., Ltd.; AST, ALT, TG, TC, MDA, SOD, glucose assay kit, insulin test kit, Masson staining kit were purchased from Nanjing Jiancheng Bioengineering Institute; IL-6ELISA kit, TNF - αELISA kits were purchased from eBioscience; hematoxylin and eosin (H&E) staining kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; Reagent, DIRECTTM kits were purchased from Lifetechnology Company of the United States; ViiA ^TM 7 real-time fluorescent quantitative PCR system was purchased from Applied Biosystems Company of the United States; III reverse transcriptase was purchased from Invitrogen Company of the United States; Accu-Chek Active blood glucose meter was purchased from Roche Company of the United States; Leica RM2255-automatic rotary microtome was purchased from Leica Microsystems (Shanghai) Trading Co., Ltd.; HistoStar ^TM tissue embedding machine Purchased from Thermo Fisher Scientific (China) Co., Ltd.; OLYMPUS BX51 optical microscope was purchased from Olympus (China) Co., Ltd.

4. Test method

(1) Establishment of high-fat diet-induced non-alcoholic steatohepatitis mouse model

Take male C57 mice and divide them into 6 groups. The blank group (Control group or CTR group) is given normal feed, the model group is given high-fat and high-cholesterol feed to induce non-alcoholic steatohepatitis, and the model group is divided into model control group (Model group), metformin (150mg/kg) group (MET group), Cyclocarya paliurus leaf total flavonoids high (CPF-1), middle (CPF-2), low (CPF-3) dosage group (500mg/kg, 250mg/kg, 125mg/kg), each group was fed for 16 weeks, administered by intragastric administration, and the blank control group and the model control group were given the same amount of normal saline.

(2) Determination of biochemical indicators

At the end of the 16th week, the tail tip blood of the mice was collected to measure the fasting blood glucose and insulin levels. After the mice were anesthetized with pentobarbital sodium, the abdominal aortic blood was collected, centrifuged at 3500rpm to prepare serum, and the levels of AST, ALT, TC, TG, IL-6, TNF-α were measured, and the liver tissue was prepared with iced PBS buffer. 10% homogenate, measure SOD, MDA level.

(3) Liver pathological examination

After mice were anesthetized with pentobarbital sodium, the liver, kidney, brain, spleen, testis, perirenal fat, and epididymis fat were removed and weighed to calculate the visceral index. Part of the liver was cut and soaked in 4% paraformaldehyde, fixed, dehydrated, transparent, embedded in paraffin, and sectioned. H&E and Masson staining were performed according to the instructions of the H&E and Masson staining kits respectively, and the pathological changes of the liver were observed.

(4) Effect of total flavonoids of Cyclocarya paliurus leaves on the mRNA level of non-alcoholic steatohepatitis

use The total RNA in the sample was extracted by the reagent, and the After the mRNA was purified by the mRNA DIRECTTM kit, the III Reverse transcriptase was used for reverse transcription, and ViiA ^TM 7 real-time fluorescent quantitative PCR system was used to detect the relevant mRNA levels in samples of each group. The primer sequence of mouse RT-PCR gene is shown in Table 1. Normal animal tissue was used as a negative control, and Gapdh was used as an internal reference. Relative quantitative analysis was carried out by 2^-ΔΔCT method.

Table 1 mouse RT-PCR gene primer sequence

(5) Statistical methods

The data are expressed as Mean ± SD, and the data are processed statistically using IBM SPSS Statistics 24.0 software. One-way analysis of variance was used to compare the means of multiple groups of samples, Dunnett's test was used for homogeneity of variance, and non-parametric test was used for heterogeneity of variance. The means of independent samples in the two groups were compared using the Independent Sample T Test, and P<0.05 was considered statistically significant.

5. Test results:

(1) Effects of total flavonoids from Cyclocarya paliurus leaves on organ indexes of NASH model mice

The body weight, liver, brain, kidney, heart, spleen, testis, epididymal fat, and perirenal fat of C57 BL/6 mice were weighed, and the organ index was calculated. It can be seen from the results that the total flavonoids of Cyclocarya paliurus leaves can control the weight gain of C57 BL/6 mice induced by high-fat and high-cholesterol diet, and at the same time reduce the liver/body weight % ratio, showing the effect of protecting the liver, and presenting a dose-dependent effect. relation. B-ultrasound results showed that liver and kidney echoes were enhanced, indicating fat accumulation, and after the intervention of total flavonoids from Cyclocarya paliurus leaves, B-ultrasound results tended to normal values. The total flavonoids of Cyclocarya paliurus leaves also has a protective effect on the brain, spleen, testis and other organs of non-alcoholic steatohepatitis model mice, making the indicators of these organs tend to the normal feeding group. In addition, the total flavonoids of Cyclocarya paliurus leaves can reduce the accumulation of fat around the kidneys and epididymis in the abdomen, inhibit fat formation and control body weight, and present a dose-effect relationship. The results are shown in Figure 2.

(2) Effects of total flavonoids from Cyclocarya paliurus leaves on biochemical indicators in mice with nonalcoholic steatohepatitis

The levels of serum transaminase AST, ALT, inflammatory factors IL-6, TNF-α, blood lipid indexes TG, TC, and lipid peroxidation index MDA in nonalcoholic steatohepatitis model mice were significantly increased, and the level of oxidative stress index SOD was significantly increased. then dropped significantly. After the intervention of total flavonoids from Cyclocarya paliurus leaves, it can reduce the levels of transaminases, inflammatory factors and blood lipids, improve the way of lipid peroxidation, and present a dose-effect relationship. The results are shown in Figure 3.

(3) Effects of total flavonoids from Cyclocarya paliurus leaves on liver pathology in NASH model mice

After C57 mice were fed with high-fat and high-cholesterol diet for 16 weeks, the appearance of the liver was earthy yellow, which was significantly different from the bright red of normal liver. The liver morphology and color of the C57 BL/6 mice treated with the total flavonoids of Cyclocarya paliurus leaves tended to be consistent with the livers of the normal control mice, the results are shown in Figure 4A.

H&E results showed that the liver tissue of mice in the Control group was evenly colored, the hepatocyte cords were arranged regularly, and the liver sinusoids were clearly visible. The liver tissue lobule structure of the mice in the model group was still intact, the liver plates were still neatly arranged, the liver cells were obviously swollen, and there were mixed large and small vesicles. Hepatic steatosis, filled with a large number of fat vacuoles of different sizes, especially around the portal area, and a large number of hepatocytes with slightly larger volume and darker nuclear staining; a large number of dots and focal necrosis in the lobules; a large number of small bile ducts can be seen Hyperplasia; portal area expanded, a large number of lymphocytes infiltrated, and inflammatory cell infiltration was obvious. In the liver tissue of mice in the high, medium and low dose groups of total flavonoids of Cyclocarya paliurus leaves, the lobule structure was complete, the liver plates were arranged neatly, the fat vacuoles were significantly reduced, and the central vein was the center, and gradually improved, and the cord structure basically existed. The tube area was slightly expanded, and inflammatory reactions still occurred in the local area, but the infiltration situation improved. The results are shown in Figure 4B, and the scoring results of each group of liver H&E staining cases are shown in Table 2. The results of silver staining and Masson staining showed that the fibrosis of the portal area in the liver of the mice in the model group was enlarged, and the formation of chronic awn-like fibers and fibrous septa could be seen. The degree of fibrosis in the portal area of the liver tissue of the mice in the high, medium and low dose groups of total flavonoids from Cyclocarya paliurus leaves was reduced, and the awn-shaped fibers were reduced. The results are shown in Figure 4C. It is proved that the total flavonoids of Cyclocarya paliurus leaves can protect the liver by inhibiting the accumulation of fat in the liver and inhibiting the fibrosis of liver cells.

Table 2 Scores of liver H&E staining cases

group fatty degeneration balloon burst inflammation control 0 0 0 model 2.4±0.52 2.9±0.32 2.7±0.48 MET 1.5±0.53** 2.0±0.47*** 1.7±0.67** CPF1 2.3±0.48 2.4±0.70 2.4±0.52 CPF2 1.8±0.63* 2.1±0.57** 2.1±0.74* CPF3 1.7±0.48** 1.8±0.42*** 1.8±0.63**

(4) Effects of total flavonoids from Cyclocarya paliurus leaves on lipid metabolism-related pathways in NASH model mice

In order to investigate the effect of total flavonoids from Cyclocarya paliurus leaves on liver lipid metabolism, and reveal the mechanism of reducing liver lipid deposition by total flavonoids from Cyclocarya paliurus leaves, the present invention investigated the expression levels of genes related to lipid metabolism. In the NASH model, 9 enzymes closely related to lipid metabolism were detected by RT-PCR. The results of RT-PCR showed that compared with normal mice, the expressions of LPL, CYP7A1, CYP27A1, PPARγ, HMGCR, CD36, aP2 and FXR in the liver tissue of mice in the NASH model were all increased, while the total flavonoids of Cyclocarya paliurus leaves After treatment, the expression levels of the above genes were significantly reduced, and the specific results are shown in FIG. 5 . It is speculated that the total flavonoids of Cyclocarya paliurus leaves interferes with the process of NASH by inhibiting the lipid production in the liver and promoting the metabolism of triglycerides.

PPARγ, FXR, and LXRα are the transcription factors of liver lipid production, and HMG-CoA reductase is the rate-limiting enzyme of cholesterol synthesis, and their mRNA levels are significantly increased (P<0.001). The gene expression level of the key lipase was also significantly increased. As an important enzyme of liver cholesterol catabolism and excretion, the mRNA levels of cholesterol 7α-hydroxylase (CYP7A1) and cholesterol 27α hydroxylase were significantly increased, and triglyceride The mRNA level of lipoprotein lipase (LPL), the rate-limiting enzyme hydrolyzed into fatty acids, was also significantly increased, and the increased mRNA level was reversed after the intervention of total flavonoids from Cyclocarya paliurus leaves.

The gene expression level of fatty acid translocase CD36/FAT related to lipid and lipoprotein transport in the model group was significantly higher than that in the control group, therefore, more free fatty acids and cholesteryl esters (CE) were transported to the liver through CD36/FAT. Cellular transport and accumulation in the liver as lipid droplets.

It can be seen from the above results that the total flavonoids of Cyclocarya paliurus leaves interferes with the process of NASH by inhibiting liver lipid production, promoting triglycerides, inhibiting fat accumulation in the liver, and promoting cholesterol catabolism and excretion.

Claims (6)

1. a kind of preparation method of Qingqian Willow leaf general flavone, which comprises the following steps:

A. the dried leaf for taking blue or green money willow, is crushed, and is crossed 40-50 mesh, is obtained blue or green money willow coarse powder, blue or green money willow coarse powder is placed in ethyl alcohol 0.5~1.5h is impregnated in aqueous solution, reheats 1~3h of refluxing extraction, is filtered merging after extracting 3-5 times altogether, is obtained blue or green money willow Leaf extract；

B. Qingqian Willow leaf extracting solution made from step a is added into polyethylene glycol and phosphate buffer, forms double-aqueous phase system；

C. the pH value for adjusting double-aqueous phase system is 6.5-7.5, magnetic agitation 10min, then stands 1-2h under the conditions of 25 DEG C, green Money willow leaf general flavone is enriched in phase, and glassy yellow is presented, and upper layer is the mixing of Qingqian Willow leaf general flavone solution and polyethylene glycol Object；

D. the upper layer mixture that removing step c is obtained, upper D101 macroporous resin column remove polyethylene glycol with ultrapure water elution, then It is eluted with ethanol water, obtains Qingqian Willow leaf general flavone eluent；

E. the resulting Qingqian Willow leaf general flavone eluent of step d further depressurized, be concentrated, it is dry to get.

2. the preparation method of Qingqian Willow leaf general flavone as described in claim 1, which is characterized in that the ethyl alcohol in the step a The quality of aqueous solution is 5-15 times of blue or green money willow meal quality, and the volume fraction of ethanol water is 55~95%.

3. the preparation method of Qingqian Willow leaf general flavone as described in claim 1, which is characterized in that poly- second two in the step b The mass concentration range of alcohol is 15%-25%；The mass concentration range of the phosphate buffer is 15%-20%.

4. the preparation method of Qingqian Willow leaf general flavone as described in claim 1, which is characterized in that the blue or green money willow of the step c The concentration of leaf flavonoids solution is 50mg/mL.

5. the preparation method of Qingqian Willow leaf general flavone as described in claim 1, which is characterized in that the D101 of the step d is big In the resin column of hole, applied sample amount and the D101 macroreticular resin volume ratio are 1: 15, and the volume of the ultrapure water is that the D101 is big 20 times of hole resin column volume, the volume fraction of the ethanol water are 80%, and volume is the D101 macroreticular resin cylinder Long-pending 15 times.

6. Qingqian Willow leaf general flavone made from the preparation method of Qingqian Willow leaf general flavone according to claim 1-5 Application in the drug or health care product that preparation prevents and treats nonalcoholic steatohepatitis.